P: 57 Thrombospondin-1 Worsens Azoxymethane-Induced Hepatic Encephalopathy Through Activation of Transforming Growth Factor Beta 1

Abstract

BACKGROUND: Acute liver failure (ALF) is a consequence of severe hepatic injury and is associated with poor clinical outcomes. Patients with ALF often present with neurological complications, called hepatic encephalopathy (HE). Transforming growth factor beta 1 (TGFβ1) is upregulated following liver damage and we have shown that TGFβ1 drives HE progression. Thrombospondin-1 (TSP-1) can activate latent TGFβ1 and therefore, we hypothesize that hepatic-derived TGFβ1 is activated by TSP-1, which exacerbates liver damage and HE associated with azoxymethane-induced ALF. METHODS: Male C57Bl/6 mice were treated with azoxymethane (AOM; 100 mg/kg BW) to induce ALF. Six hours after AOM injection, mice were injected with the TSP-1 antagonist LSKL (30 mg/kg) or SLLK as control. In parallel, male TSP-1 knockout mice and wild-type (WT) controls were injected with AOM. In AOM-treated mice, cognitive impairment was monitored by reflex and ataxia measurement. Liver histology was assessed by hematoxylin and eosin staining and serum transaminases were measured. Cleaved caspase 3 immunohistochemistry and TUNEL staining were used to assess apoptosis in liver tissue. Hepatic inflammation was determined by measuring IL-1β, IL-6 and TNFα expression via real-time PCR and ELISA assays. TGFβ1 and TSP-1 expression were assessed in liver, serum and cortex by immunoblotting, immunohistochemistry and real-time PCR. Cerebral edema and microglia activation were assessed and neuroinflammation was measured by assessing IL-1β, IL-6 and TNFα expression in the cortex. RESULTS: Mice injected with AOM had elevated hepatic, circulating and cortical TGFβ1 and TSP-1 levels, with the cortex only having elevated TGFβ1. LSKL-treated mice and TSP-1 knockout mice administered AOM had reduced activation of hepatic TGFβ1, hepatocyte apoptosis, inflammation, and hepatic injury compared to AOM and SLLK-treated mice or WT AOM-treated mice. LSKL-treated and TSP-1 knockout mice administered AOM had an increased latency to reach coma compared to SLLK-treated or WT mice. LSKL-treated mice and TSP-1 knockout mice had reduced TGFβ1 expression, less cerebral edema, attenuated microglia activation, and decreased expression of IL-1β, IL-6 and TNFα in the cortex compared to control mice. CONCLUSIONS: TGFβ1 and TSP-1 were elevated in the livers and serum of AOM-treated mice and strategies employed to reduce TSP-1 signaling reduced liver damage and neuroinflammation in the AOM mouse model of HE. Therefore, targeting TSP-1 signaling may be a novel therapeutic target for the management of both ALF and HE following acute liver injury.